Summary/Abstract ? Project 2 The long-term objective of this project is to investigate how post-transcriptional gene regulatory mechanisms tilt the interaction of herpes simplex virus (HSV) with neurons either towards lytic infection or towards latency. HSV latency is the most fascinating biological property of the virus and its most important clinical feature. Understanding HSV latency may lead to new therapies or even a cure for this widespread pathogen. The first specific aim of this project is to investigate repression of lytic gene expression during latency. At least one microRNA (miRNA), host miR-138, represses lytic gene expression and promotes HSV latency, but much remains unknown about how this or other miRNAs impact HSV infections. The roles of miR-138, miRNAs more generally, and miRNAs from the latency associated transcript (LAT) locus will be investigated using mice whose miR-138 or Dicer genes can be inducibly excised in sensory neurons. Effects of such conditional knockouts on viral replication and reactivation, viral gene expression, chromatin status, and latency will be measured in vivo and, in collaboration with Projects 1 and 3, in cultured neurons. Two specific hypotheses regarding how products of the LAT locus repress ICP4 gene expression will be tested. With Project 1, a hypothesis regarding transcription antisense to the ICP4 gene or the corresponding transcripts will be tested using viral mutants that should exhibit decreases in such transcription. The hypothesis that miR-H6 represses ICP4 expression will be tested using mutants with disrupted miR-H6 expression or binding sites for it. The second specific aim focuses on post-transcriptional ? most likely translational ? mechanisms restraining expression of the viral protein ICP34.5 that counteracts host immunity. How mutations affecting the 5' untranslated region of ICP34.5 mRNA increase ICP34.5 expression and viral virulence will be studied, and, with Project 3, their effects on immune mechanisms will be assessed. The third specific aim assesses a post- transcriptional mechanism that may tilt the balance towards lytic infection. How HSV-1 blocks miRNA biogenesis by preventing export of miRNAs from the nucleus during lytic infection, which may overcome repressive functions of latent miRNAs, will be studied. The viral gene product(s) responsible will be identified by testing HSV-1 open reading frames from Project 1 and viral miRNAs for blocking pre-miRNA to miRNA conversion in miRNA-transduced cells, and by testing viral mutants. How the gene product(s) cause this blockade will be investigated. The fourth aim seeks to identify targets for miRNAs from the LAT locus by using deep sequencing based methods. Candidate targets will be tested for their roles in HSV replication and other biological activities in collaboration with Projects 1 and 3. Throughout this project, studies of gene expression and chromatin status will be assisted by Core A, and studies using mice will be assisted by Core B.